Scientists within the Laboratory of Genomic Integrity (LGI) study the mechanisms by which mutations are introduced into damaged DNA. It is now known that many of the proteins long implicated in the mutagenic process are, in fact, low-fidelity DNA polymerases that can traverse damaged DNA in a process termed translesion DNA synthesis (TLS). The TLS polymerases gain access to a nascent primer terminus via an interaction with the cells replicative, ring-shaped, clamp (beta-clamp in E.coli and PCNA in eukaryotes). The process is initiated by a clamp loader (gamma-complex in E.coli and replication factor C in eukaryotes), which recognizes the DNA primer terminus and opens and assembles the clamp around the nascent DNA. Each clamp has two (prokaryotes), or three (eukaryotes) potential DNA polymerase binding sites and may, therefore, engage multiple polymerases simultaneously. Indeed, such interactions are believed to be critical for switching between replicative and TLS polymerases. In vitro studies investigating the effects of the replicative clamps on TLS have been hampered because the clamps readily slide off of linear DNA substrates. One option is to cap the DNA ends using large biomolecules such as Streptavidin beads linked to biotinylated oligonucleotides. However, this imposes large steric constraints and may affect the ability of the DNA polymerase to access the primer terminus. Circular, single-stranded templates are, therefore, more likely to provide more informative data on the effects of the replicative clamps on TLS and polymerase switching in vitro. We have therefore developed a protocol for the rapid and efficient purification of circular, single-stranded DNA containing a defined lesion. To achieve our goal, we used a primer containing a site-specific DNA lesion and annealed it to a single-stranded DNA template containing Uracil. After primer extension and ligation, the double-stranded DNA was degraded in vitro using the combined actions of E.coli Uracil DNA glycosylase and Exonucleases I and III. The final product is a circular, single-stranded DNA molecule containing a defined lesion that can be used for in vitro replication and repair assays. Most damage-induced (SOS) mutagenesis in Escherichia coli occurs when DNA polymerase V, activated by a RecA nucleoprotein filament (RecA*), catalyzes TLS. The biological functions of RecA* in homologous recombination and in mediating LexA and UmuD cleavage during the SOS response are well understood. In contrast, the biochemical role of RecA* in pol V-dependent mutagenic TLS remains poorly characterized. Proposals for the role of RecA* in TLS have evolved from positioning UmuD'C on primer/template DNA proximal to a lesion, to a dynamic interaction involving displacement of RecA* filaments on the template by an advancing pol V, to a model in which RecA* need not be located in cis on the template strand being copied, but can instead assemble on a separate ssDNA strand to transactivate pol V for TLS. As part of a collaborative study with Myron Goodman (University of Southern California), we addressed the hitherto enigmatic role of RecA* in polV-dependent SOS mutagenesis. We demonstrated that RecA* transfers a single RecAATP stoichiometrically from its DNA 3'-end to free pol V (UmuD'2C) to form an active mutasome (pol VMut) with the composition UmuD'CRecAATP. Pol VMut catalyzes TLS in the absence of RecA* and deactivates rapidly upon dissociation from DNA. Deactivation occurs more slowly in the absence of DNA synthesis, while retaining RecAATP in the complex. Reactivation of pol VMut is triggered by replacement of RecAATP from RecA*. Thus, the principal role of RecA* in SOS mutagenesis is to transfer RecAATP to pol V, so as to generate active mutasomal complex for translesion synthesis. Human cells posses at least 14 DNA polymerases (pols). Three, pols alpha, delta and epsilon are involved in genome duplication. The remaining eleven DNA polymerases have specialized functions within the cell. Four of the specialized DNA polymerases (pols eta, iota and kappa and Rev1) belong to the Y-family of DNA polymerases and participate in TLS. Unlike cellular replicases, which are endowed with high processivity, high catalytic efficiency and high fidelity, Y-family TLS DNA polymerases exhibit low processivity, low catalytic efficiency and low fidelity. To facilitate the ongoing studies of the enzymology and cellular roles of these polymerases, a robust and flexible method for monitoring their catalytic activity is needed. In a collaborative study with Anton Simeonovs group (NHGRI), we developed a fluorescence-based assay to study the enzymology of TLS DNA polymerases in real time. The method is based on a fluorescent reporter strand displacement from a tripartite substrate containing a quencher-labeled template strand, an unlabeled primer, and a fluorophore-labeled reporter. With this method, we could follow the activity of human DNA polymerases eta, iota and kappa under different reaction conditions. Last, but not least, we demonstrated that the method can be used for small molecule inhibitor discovery and investigation in highly miniaturized settings and we reported the first nanomolar inhibitors of Y-family DNA polymerases iota and eta. We hypothesize that the fluorogenic replication assays described above should facilitate further mechanistic and inhibitor investigations of the TLS DNA polymerases.